The vertebrate immune system is characterized by its ability to respond to an enormously diverse set of antigenic determinants, or epitopes. This response is effected through T and B lymphocytes, commonly referred to as T cells and B cells. The immune system, comprising these specialized cells, recognizes and processes foreign pathogens and macromolecules, typically clearing them from the body. Lymphocytes individually exhibit high specificity in recognition of particular molecular shapes and collectively exhibit great diversity in reacting with, i.e., specifically binding, a broad range of molecular structures. These properties are mediated through both T-cell receptors and immunoglobulins that serve as antigen receptors.
T cells usually recognize only antigens that are present on the surfaces of other cells. This recognition event generally occurs only when the antigens are correctly presented in combination with polymorphic cell surface molecules encoded by the major histocompatibility complex (MHC). These T cells can be subdivided into separate functional categories including cytotoxic effector cells (T.sub.c), inducer or helper cells (T.sub.h) and suppressor cells (T.sub.s).
T cells mature in the thymus, in contrast to B cells which differentiate in the fetal liver or bone marrow. Among the cell surface molecules which characterize T cells are the .alpha. and .beta. chains of the T-cell receptor molecule. General introductory review articles describing the T-cell receptor include Toyonaga and Mak, (1987) "Genes of the T-Cell Antigen Receptor in Normal and Malignant T-Cells" in Ann. Rev. of Immunology 5:585-620; Kronenberg et al., (1986) "The Molecular Genetics of the T-Cell Antigen Receptor and T-Cell Antigen Recognition" in Ann. Rev. of Immunology 4:529-591; and Davis, (1985) "Molecular Genetics of the T-Cell Receptor Beta Chain" in Ann. Rev. of Immunology 3:537-560, each of which is incorporated herein by reference.
T-cell antigen receptor polypeptides are members of the immunoglobulin superfamily. Members of the immunoglobulin superfamily are characterized by similarities in primary and tertiary structure. They also display characteristic gene segment organization, ontogeny of expression and diversification. In particular, the polypeptide chains of the immunoglobulins typically result from the rearrangement, at the genetic level, of various variable region (V), joining region (J), and constant regions (C) gene segments, or V, diversity region (D), J and C gene segments. Although particular chains may have different sized repertoires of the various segments, the genetic rearrangements and combinatorial joining and junctional diversity all contribute in a characteristic manner to produce the extraordinary diversity of polypeptide sequences exhibited by these gene families. See, Hood et al., (1985) Cell 40:225-22, which is incorporated herein by reference.
A T-cell antigen receptor molecule comprises two polypeptide chains, generally an alpha chain and a beta chain, each of which exhibits two extracellular immunoglobulin-like domains. Such molecules comprise an amino terminal variable region domain (V) and a carboxy terminal constant region domain (C), more specifically designated with .alpha. or .beta. when indicating the particular chain of origin. Each of these domains is normally stabilized by a disulfide bond between two conserved cysteine residue pairs on each chain. The two chains are apparently attached to one another by an inter-chain disulfide bond near the cell membrane outer surface. Each chain is anchored on the membrane by a hydrophobic transmembrane segment which typically spans the entire membrane lipid bilayer. A short carboxy terminal segment extends into the cytoplasm. Both alpha and beta chains are normally N-glycosylated at sites near the disulfide bonds and at a site near the extracellular surface of the cell membrane.
The alpha and beta chains of the T-cell receptor are encoded by gene segments analogous to the variable region (V) segment, the joining region (J) segments, the diversity (D) segment, and the constant region (C) segments of immunoglobulin genes. Diversity in the T-cell receptor repertoire arises, in part, from the rearrangement of V, D, and J gene segments and from the insertion or deletion of nucleotides at the V.sub..beta. -D and D-J.sub.62 or V.sub..alpha. -J.sub..alpha. junctions. Some cells express T-cell receptor gamma and delta chains in place of the alpha and beta chains.
Although specimens of alpha and beta chains of T-cell antigen receptors from mouse and from human have been isolated, it has been difficult to raise antibodies of desired specificity against these polypeptides. T-cell receptors are expressed at very low levels on T lymphocytes, with conventional biochemical purification generally being unfeasible. Thus, physical characterization studies have been almost non-existent. In general, the production of antibodies against native T-cell receptors has also been unproductive.
Previous workers have reported solubilization of the T-cell antigen receptor (TCR) V.sub..alpha. domain as a chimeric molecule in either the form V.sub..alpha. -C.sub.H1 -H-C.sub.H2 -C.sub.H3 or the form V.alpha.C.kappa., where C.sub.H1 -H-C.sub.H2 -C.sub.H3 and C.sub..kappa. are, respectively, immunoglobulin heavy and light constant regions. See Gascoigne et al., (1987) Proc. Natl. Acad. Sci. USA 84:2936-2940; Mariuzza and Winter, (1989) J. Biol. Chem. 264:7310-7316). Gascoigne et al. reported solubilizing the chimeric V.sub..alpha. -heavy chain in conjunction with a mouse .lambda. light chain, whereas Mariuzza and Winter reported solubilizing the chimeric V.sub..alpha. C.sub..kappa. light chain homodimers expressed in the absence of heavy chain expression.
Consistently obtaining highly efficient, secreted expression of substantially full length T-cell receptor polypeptide segments remains a major research goal. Although some genetic fusions between T-cell receptor domains and immunoglobulin segments have been constructed, the expression products typically have resulted in insoluble polypeptides. Thus, there exists a need for improved methods for producing large quantities of water-soluble T-cell receptor analogues which will permit the efficient production of antibodies against natural T-cell receptor epitopes. Preferably, when presented to target immune systems the analogues will not induce a significant number of antibodies against epitopes from the non-TCR segments. In particular, polypeptide segments of T-cell receptors which can be effectively reconstituted into conformations highly analogous to their native state would be most preferred.
Furthermore, the absence of a full repertoire of reagents for distinguishing the various T-cell receptor family types has hindered progress in the understanding and treatment of many immunological disorders. The present invention answers these and other needs.